Bionanotechnology is a multidisciplinary area of knowledge that combines biological principles with physical and chemical procedures to research and develop nano-level materials with specific functions and new properties. Nanomedicine is the application of bionanotechnology in the field of biomedical sciences and has become a fundamental tool for development of new drug products. One of the biggest challenges in this area is to reduce or eliminate the immune response during nanomaterial supply, as well as to improve its effect by lengthening its bioavailability in the body.
Cancer is a global public health problem that has not yet been resolved affecting health of more than 14 million people annually, 50% of whom die [1]. The most used treatment to fight this important disease is chemotherapy, which in many cases is successful but has dramatic side effects. These side effects can put the patient at risk and can lead to death. Classic drugs used in chemotherapy are mitotic inhibitors, alkylating agents, antimetabolites, topoisomerase inhibitors and anthracenediones (anthracyclines) that inhibit DNA synthesis and mitosis, in order to prevent the rapid proliferation of cells. However, these agents are substances that can exert their toxic activity in healthy cells. Hence the unwanted side effects [2].
Most drugs used in chemotherapy must be activated by P450 cytochromes (CYP) to exert their antitumor activity [3, 4] (Table 1).
TABLE 1Examples of commercial antitumor prodrugs activatedby human CYP (modified from Huttunen et al., 2008 [7]).Human CYPActivationcatalyzing theProdrugActive drugmechanismreactionCyclofosfamideFosfamideHydroxylationCYP286, CYP2C9,mustardCYP3A4IfosfamideIfosfamideHydroxylationCYP2B6, CYP3A4mustardTrofosfamideTrofosfamideHydroxylationCYP2A6, CYP2B6,mustardCYP2C9, CYP3A4PradefovirPMEA-HydroxylationCYP3A4triphosphateMB07133araC-HydroxylationCYP3A4triphosphateMB07811MB07344HydroxylationCYP3A4BuparvaquoneBuparvaquoneOxidationCYP3A4, CYP2BhydroxyimineNabumetoneNabumetoneOxidationCYP3A4, CYP2A6hydroxyimineDB289FuramidineO-demethylationCYP4F(DB75)reductionSibrafibanRo 48-3888Reduction—XimelagratanMelagartanReduction—GuanoxabenzGuanabenzReduction—AQ4NAQ4ReductionCYP3A4, CYP1A1,CYP1A2, CYP2B6DacarbazineMTICHydroxylationCYP1A1, CYP1A2,(DTIC)CYP2E1Tegafur5-FUHydroxylationCYP2A6, CYP1A2,CYP2C84-ipomeanolOxidationCYP1A2, CYP2B7,(epoxidation)CYP2C19,CYP2D6, CYP2F1,CYP3A3, CYP3A4,CYP4B1DDMX (PNU-PNU-159682CyclationCYP3A4152243)Tamoxifen4-Hydroxy-HydroxylationCYP2D6tamoxifenTamoxifenN-Demethyl-N-DemethylationCYP1A1, CYP1A2,tamoxifenCYP1B1, CYP2C9,CYP2C19,CYP2D6, CYP3A4,CYP3A5TamoxifenEndoxifenHydroxylation,CYP2D6, CYP3A4N-DemethylationClopidogrelR-130964OxidationCYP3A4, CYP3A5
Due to its activity on certain prodrugs (mainly antiviral drugs and chemotherapy drugs) P450 cytochromes (CYPs) are important because is the largest enzymatic complex involved in drug metabolism in our body, playing a key role in metabolism oxidative phase.
CYPs form a large family of microsomal hemoproteins that catalyze many types of oxidation reactions on endogenous and exogenous substrates [7]. Table 1 shows some of the commercially available drugs that are activated by CYPs. There are more than 270 families of different CYPs of which 18 have been identified in mammals. Humans have between 55 and 60 genes that code for CYP, which are expressed mainly in liver and intestines, but may be found in almost all tissues. At cell level they are found in endoplasmic reticulum membranes. In general, CYPs that metabolize endogenous compounds are very specific for certain substrates, while CYPs that metabolize exogenous compounds show a low specificity and are capable of transforming a large variety of compounds [7].
Reactions catalyzed by CYPs are based on monooxygenation, where one atom of the oxygen molecule is incorporated into the substrate. Generally, these enzymes are part of a multienzyme complex since oxygen molecule (O2) activation and the consequent transfer of an oxygen atom to the substrate involves the transfer of electrons from NADPH to CYP, facilitated by other proteins such as cytochrome P450 reductase.
However, the activity of these enzymes in different tissues varies significantly [5], and even more, in some cases, as in breast cancer, CYP activity is much lower in the tumor than in the surrounding healthy cells [6]. This hinders drug supply and dosage, also inducing cytotoxicity in healthy tissues.
On the contrary, CYPBM3 is, for example, a bacterial-origin protein, structurally and functionally similar to human microsomal cytochromes [7b, 7c], with the advantage of being a soluble and stable CYP in aqueous medium, further being able to be produced in large quantities unlike human CYPs. Another interesting feature of this CYP is that it has high plasticity to be subject to both site-directed mutagenesis and directed evolution, to obtain variables capable of transforming a wide variety of non-natural substrates such as alkanes, polyaromatic hydrocarbons and medications [7d; 7e]. These properties make CYPBM3 a versatile enzyme with a series of interesting operational advantages to be used as a model enzyme for encapsulation in viral nanostructures.
Drug Activation Therapy Through Enzymes
Prodrug activation therapy using exogenous enzymes is a proposed strategy to increase efficiency of certain medical procedures, such as chemotherapy. Chemotherapy being one of the most used treatments nowadays to fight cancer [8] shows drastic side effects. One of the objectives of present patent is to increase the local concentration of active drug in tumor cells, which would increase the drug efficiency in the tumor and reduce the toxicity produced by the drug in the rest of the host cells [9]. This strategy is carried out in two steps; firstly, the exogenous enzyme must be directed to the cells of interest and accumulated in the site, then, in a second step, the prodrug (either directed or systemically) is administered, selectively activating in the target cells.
Two methods have been proposed, broadly speaking, to carry out an enzyme supply capable of activating the prodrug, to the cells of interest: gene delivery, known by its acronym in English as GDEPT and active enzyme supply [10].
Gene therapy has been proposed as an alternative to increase CYP activity in tumor cells. GDEPT (gene-directed enzyme prodrug therapy) technique involves introduction to specific tumor cells, of one or several genes that encode for enzymes with the ability to transform prodrugs. These genes can be delivered to tumor cells using different vectors, such as those of viral type [10]. On the other hand, antibody-based therapy, known as ADEPT (antibody-directed enzyme prodrug therapy) has been one of the most developed therapies. The enzyme capable of transforming the prodrug selectively targets target cells, through conjugation with an antibody, which has the ability to specifically bind to antigens that are expressed on tumor cell surface [11]. For both strategies, GDEPT and ADEPT, enzyme-prodrug systems have been developed that have been tested in clinical trials [12,13]; However, there is still no treatment in use based on these therapies to date.
A promising alternative to overcome the problems associated with gene insertion and expression in human (mammalian) cells, is to directly deliver the enzyme to specific targets through virus-like particles (VLPs),
Viral Capsids
Viral capsids or “virus-like particles” (VLPs) are composed only of viral layer proteins and unlike viruses, do not contain the natural genetic material thereof, so they are not infectious particles. These particles can be used as basic scaffolds for nanostructured material design and manufacture. Within this context, some of the features that make VLPs attractive are the following [17, 18]:
i) highly ordered architectures of nanometric dimensions that have the ability to self-assemble;
ii) about 1031 viruses are estimated to inhabit the Earth [19], there is a great diversity of both sizes (17-1500 nm in icosahedral capsids) within this vast number, as well as in different forms, with icosahedral, filamentous capsids and helical forms being predominant;
iii) monodisperse structures in size and composition, under pH and ionic strength particular conditions;
iv) large surface areas, with a variety of functional groups exposed in a high number of copies, that allow coupling of multiple ligands, either of the same or different molecules. This characteristic makes them polyvalent molecules, with the ability to participate in collectively stronger interactions than their counterparts with unique interaction sites, increasing the binding affinity with the target sites;
v) have cavities that can be used to encapsulate molecules for various purposes;
vi) due to their protein nature they are biocompatible and biodegradable.
Viral nanoparticles have three available interfaces to be either chemically or genetically manipulated: the outer surface, the interface between the protein subunits and the internal surface [20]. The latter surface has been used to encapsulate various materials such as metals [21], drugs [22], DNA [23] and proteins [24] in order to generate new materials, catalysts and delivery systems. Protein encapsulation has focused mainly on the introduction of fluorescent proteins into protein nanostructures. The most widely used model has been green fluorescent protein (GFP) due to its easy detection [25, 26, 27, 28]. However, there are a number of works nowadays where enzymes have been encapsulated inside such containers, generating bionanoreactors with properties and catalytic capacities different from their non-encapsulated counterparts.
Due to its size, pseudo-viral particles cannot be filtered and eliminated by kidney (removal threshold <40 kDa), staying longer circulating in the body (increase of residence time within the body). Moreover, those particles can be modified to modulate their residence time in the bloodstream. Finally and of importance for cancer therapy, particles in the nanometric order (100-500 nm) have been observed that preferentially accumulate within solid tumors due to a phenomenon known as increased permeability and retention effect (EPR effect). This accumulation is due to tumor-promoted blood vessels surrounding said tumor, presenting a disorganized architecture with a series of holes in their structure (200-800 nm), allowing nanoparticle extravasation into the tissue. In addition to that above, particles are retained in these sites due to a deficient lymphatic drainage proper of tumors [29, 30].
For example, P22 bacteriophage is a double-stranded DNA virus that infects Salmonella typhimurium. The 58 nm icosahedral nanostructure is composed of some minority proteins (expulsion and portal proteins) and by 420 coat proteins (CP) that are assembled with the help of 60 to 300 scaffold proteins (scaffold protein, SP) in a structure known as procapsid. The P22 procapsid-derivated pseudo-viral particle only requires the capsid and scaffolding protein to be assembled. The layer protein consists of 430 amino acids (46.6 kDa) folded into eight distinct domains. In the absence of the scaffold protein, the coat protein is not assembled or, in high concentrations, forms T=4 spheres as well as spiral aberrant structures [31]. The procapsid is made up of 72 capsomeres of the coat protein, twelve of which are forming pentamers and 60 of them hexamers. These hexamers are distorted, with a pore in the center with a diameter ranging from 3 to 4.5 nm [32].
Enzyme Encapsulation Within Viral Capsids
Protein encapsulation within these viral origin vehicles offers a series of advantages to overcome protein limitations as therapeutic agents. First of all, capsids are vehicles with a high load capacity, suitable for transporting considerable protein amounts therein. In addition, the viral nanostructure is capable of conferring protein encapsulated protection against protease degradation [35, 36], as well as a barrier against immune system recognition [37]. Virus immunogenicity can be killed by different methods, such as epitope modification, “self-peptides” [38] and particle coating with polymers such as polyethylene glycol (PEG) [39]. In this way the capsid is chemically modified and not the biopharmaceutical drug in question, thus avoiding negative repercussions on the biological activity of therapeutic protein.
Enzyme encapsulation within pseudo-viral particles has been carried out mainly for the production of bionanoreactors focused on catalysis phenomena study [35, 36, 40, 41, 42, 43, 44], although its use as possible therapeutic agents has also been proposed [37]. The first article reported on enzyme encapsulation in pseudo-viral particles was in 2007, where Cornelias-Aragonés [40] et al., designed a system to study enzyme kinetic behavior at individual level, based on encapsulation of a white horseradish peroxidase in capsids derived from CCMV virus (Cowpea chlorotic mottle virus). After this first work, encapsulation of multiple enzymes (single-variety variants) in different capsids was carried out using different encapsulation strategies (Table 2). High enzyme concentrations, in millimolar amount, reached within viral capsids allowed study of catalysis phenomena in crowded environments simulating those found at cellular level, which would allow a better understanding of such biocatalyst function inside cells.
TABLE 2Multiple enzyme encapsulation of the same type inviral capsidsKcat/KmMconfEnzymesregardingEncapsulationEnzymeCapsid(nM)per capsidEfreemethodRefCytosineSV40NDNDLowerBy fusion with37deaminase(VNR)capsid internalprotein (in vivo)Peptidase FBacteriophageND 2-18Lower 3XBy fusion with35Qβ(9 enzymes)RNA (in vivo)LuciferaseBacteriophageND4-8 Lower 30XBy fusion with35Qβ(4 enzymes)RNA (in vivo)Antarctic lipase B CCMV11.3-4  HigherBy fusion with41pseudozyme(kcat)coiled-coil motif(in vitro)AlkalineBacteriophage0.53.2EquivalentBy electrostatic45phosphataseMS2(monomers)interactions.Fusion withnegative peptide(in vitro)AlcoholBacteriophage7.2249 ± 13 Lower 1.6XBy fusion with42dehydrogenaseP22scaffold protein(in vivo)CelB glycosidaseBacteriophage2.4 87 ± 3.5EquivalentBy fusion with43P22(monomers)scaffold protein(in vivo)PhosphotriesteraseBacteriophage1.140 ± 10LowerBy fusion with36P22(monomers)600Xscaffold protein(in vivo)P450 CytochromeCCMV4.931Lower 10XBy electrostatic15interactionsMconf: Confinement molarity (enzyme concentration inside the capsid).ND: Not determined.VNR: Non-reported value.
Despite finding a decrease in activity for most of encapsulated enzymes, new properties in the bionanoreactor are generated for some of these systems, such as a thermostability increase [35, 36], proteolysis resistance [35, 36], protection against the lyophilization process [36], inhibition reversal by substrate [42] and decrease to denaturation under certain operating conditions [40]. For the particular case of cytosine deaminase, which converts the 5-fluorocytosine prodrug to the 5-fluorouracil active drug, the SV40 capsid was used as a vehicle for enzymatic activity supply to CV-1 cells {cell line from monkey kidney), in order to sensitize them to prodrug treatment and induce cell death [37].
Recently, the first article was published where multiple copies of different enzymes were encapsulated in a pseudoviral particle (P22 bacteriophage). The three encapsulated enzymes, CelB glycosidase, ATP-galactosidase and ADP-glycokinase, have the peculiarity of performing a series of cascade reactions in the Pyrococcus furiosus sugar metabolism [44]. Contrary to what was expected, no increase in reaction cascade efficiency was found; it is essential to pay special attention to an adequate balance of kinematic parameters of each involved enzyme in order to design an efficient catalytic system. Construction of synthetic metabolomes based on enzyme encapsulation in pseudo-viral particles might generate complex catalytic systems with various practical applications.
Although reports of enzyme or other protein encapsulation within viral capsids to generate bionanoreactors have been disclosed, the use of VLPs as cytochrome enzyme carriers has been poorly addressed, and even with unrepresentative results. Such is the case of the CYP encapsulation in CCMV [15] managing to load up to 14 CYPs per nanoparticle.
Handling of CYPs is not trivial, besides the encapsulation described in the present invention requires the design of a strategy that included the use of a virus scaffold protein to make a fusion protein with CYP, which is not apparent even for someone with technical knowledge in the art.
CYP encapsulation offers many advantages such as those set forth in the present invention. CYPs are very unstable enzymes that lose easily their activity and are difficult to keep in active form. They are usually produced in microsomes (lipid vesicles) and cannot be stored. Being in the viral capsids, CYPs remain stable and can be used, which is very difficult with the isolated protein.
Reduction of Nanoparticle Immunogenicity
Polyethylene glycol (PEG) is an amphipathic polymer commonly used in drug supply and its basic structure is H—(O—CH2-CH2)n-OH. It is a non-immunogenic neutral molecule that can be synthesized in different lengths and has been approved by the North American Food and Drug Administration (FDA) for its use in cosmetics, foods and medicaments. There are numerous publications reporting PEG covalent binding on molecules, significantly reducing its antigenicity and immunogenicity, as well as increasing its solubility, maintaining its in vivo bioactivity [46]. Further, PEG is able to protect peptides, proteins or enzymes from degradation, increasing their survival in the body.
An example of protein immunogenicity reduction by PEG modification is trichosanthin (TCS), a protein that interacts with the type I ribosome used for AIDS and tumor treatment. Its application is limited by a very high immunogenic reaction and its residence time under bloodstream. Pegylated trichosantin has been shown to be 3 to 4 times less immunogenic and to have a non-specific toxicity 0.5 to 0.8 times lower, as well as 4.5 to 5 times longer residence time [47]. The only reported disadvantage is an activity reduction, but by having a longer in vivo circulation time, this activity reduction is compensated.
Likewise, TRAF6 protein (TNF receptor associated factor 6) is an intracellular adapter protein in the osteoclast signaling pathway. TRAF6 inhibitor peptide (SEQ ID NO. 1. DRQIKIWFQNRRMKWK) may hinder this pathway, thus avoiding excessive osteoclastic activity, but as a therapeutic agent of osteoporosis shows several limitations due to its short half-life, rapid kidney elimination, and especially its immunogenicity. However, [48] they were able to significantly improve the properties of this peptide through pegylation with a better bioavailability in laboratory animal plasma and with better incorporation at action site. Therefore, a better therapeutic agent for treatment of osteoporosis was obtained.
Finally, the recombinant human growth hormone (hGH), used in treatment of short size disorders in children and adults was modified in a specific-site way by da Silva Freitas et al. [49], showing that the two tested pegylations retained the native hGH secondary structure and also had a residence time 4.5 times higher and therefore a better systematic exposure in rat pharmacokinetics.
Functionalization and Targeting to Tumor Cells
Targeting of nanoparticles to specific tissues is studied by many research groups around the world. The process called in English “drug delivery” is a research field with promising future in medicine and pharmacology. An example of success in nanoparticle targeting to cancer cells was reported by Cai et al. [50]. Quantum dots were functionalized with a peptide (arginine-glycine-aspartic acid) to target and visualize tumor vascularization. These nanoparticles were administered intravenously in mice carrying human subcutaneous glioblastomas. Tumor luminescence showed good specificity, intensity and contrast. Subsequently, a pegylation gave better stability to quantum points. Another example is the intravenously directed delivery of DNA fragments to gliomas for gene therapy purposes [51]. This group used a highly branched dendrimer (PAMAM) on the nanoscale that was conjugated to chlorotoxin, a polypeptide that binds specifically to receptors expressed in gliomas. In this way, they were able to direct the DNA-containing nanoparticles and specifically bind to nervous system tumor cells.
In the present invention, unlike the state of the art which leads a drug in a targeted and controlled manner to a tissue or tumor in a mammalian or human patient, it is intended to bring the cytochrome P450 (cytochrome P450 enzymatic activity) to the tissue of interest with the aim of activating prodrugs in selected target cells or tissues of a mammalian or human patient; for example, that cytochrome P450 exerts its enzymatic activity in tumor cells, tumor tissues, or other tissues of interest for greater efficiency in prodrug activation, helping to contribute to cancer treatment, with a better efficiency in treatment thereof with chemotherapy in a human patient suffering from a tumor or cancer selected from breast cancer or colon cancer, or in other treatments where the prodrug is activated by cytochrome P450.
Most drugs used in chemotherapy are administered as prodrugs to a mammal or human patient. That is, they are administered in a chemical form that has no biological activity. These compounds are activated once they are ingested or injected into the body. Activation is an enzymatic or catalytic transformation mediated by cytochromes P450 that are found in different tissues.
Tamoxifen is the most widely used drug within prodrugs for treatment of hormone-dependent breast cancer [52]. Tamoxifen acts as a selective modulator of estrogen receptor, inhibiting proliferation of tumor cells [53]. This anticancer agent is metabolised by different CYP450 in the body, mainly CYP2D6 and CYP3A4, to give rise to 4-hydroxy tamoxifen and endoxifen active drugs, as well as to a number of clinically inactive metabolites [54, 55]. The active product is a very potent cytotoxic agent, so its dosage and treatment duration must be strictly controlled. The possibility of taking directly and exclusively tamoxifen to tumor cells means that necessary doses are significantly reduced, also reducing drastic side effects and increasing treatment effectiveness. On the other hand, resveratrol is a polyphenolic compound naturally produced in plants. In addition to its role as phytoalexin (antimicrobial and antioxidant activities), a series of anti-inflammatory, cardioprotective and anticancer properties have been attributed, both to prevent and treat tumor development [56]. It has been found that a hydroxylated resveratrol derivative, piceatannol, has the ability to function as a more potent chemotherapeutic agent than resveratrol among many other biological activities [57]. This compound is able to suppress cancer cell proliferation and induce apoptosis These properties make piceatannol an interesting potential drug in cancer treatment.